Simultaneous determination of delta9-tetrahydrocannabinol and 11-nor-9-carboxy-delta9-tetrahydrocannabinol in human plasma by solid-phase extraction and gas chromatography-negative ion chemical ionization-mass spectrometry.

Delta9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) in human plasma can be simultaneously detected using solid-phase extraction with gas chromatography and negative ion chemical ionization mass spectrometry. THC-d3 and THCA-d3 are added as internal standards; protein is precipitated with acetonitrile and the resulting supernatants diluted with 0.1 M sodium acetate (pH 7.0) prior to application to the solid-phase extraction columns. THC and THCA were eluted separately and then pooled, dried under air, and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatized THC-d0 gives abundant molecular anions (m/z 410), and the derivatized THCA-d0 gives abundant fragment ions (m/z 422) formed by loss of (CF3)2CHOH from its molecular anion. The recoveries of THC and THCA were 74% and 17%, respectively. The lower and upper limits of quantitation were 0.5 and 100 ng/mL for THC and 2.5 ng/mL and 100 ng/mL for THCA. The within-run accuracy and precision for THC (measured at 0.5, 1, 10 and 75 ng/mL) ranged from 98 to 106% (% target) and 4.1 to 9.5 (%CV), respectively. For THCA, the within-run accuracy and precision (measured at 2.5, 5, 10, and 75 ng/mL) ranged from 89 to 101% and 4.3 to 7.5%, respectively. The between-run accuracy and precision for THC ranged from 92 to 110% and 0.4 to 12.4%, respectively. The between-run accuracy and precision for THCA ranged from 97 to 103% and 6.5 to 12.3%, respectively. In processed samples stored in reconstituted form at -20 degrees C, THC and THCA were stable for at least three days. THC and THCA stored in plasma were stable following three freeze/thaw cycles. THC and THCA in whole blood at room temperature for 6 h, or in plasma stored at room temperature for 24 h, did not show significant change. Storage in polypropylene containers for 7 days at -20 degrees C and the presence of 1% sodium fluoride or the cannabinoid receptor antagonist, SR141716, at 1 microg/mL did not interfere with the quantitation of THC and THCA. In three individuals who smoked marijuana under controlled dosing conditions, peak THC concentrations of 151, 266, and 99 ng/mL were seen in the first plasma samples drawn immediately after the end of smoking, and corresponding peak THCA concentrations of 41, 52, and 17 ng/mL occurred at 0.33 to 1 h after cessation of smoking.

Drug quantitation on a benchtop liquid chromatography-tandem mass spectrometry system.

The specificity and selectivity of LC-MS-MS is illustrated to explain why LC-MS-MS has become the method of choice for quantitation within the pharmaceutical industry. Two assays are described that demonstrate the facility with which new ion trap technology can utilize the selectivity and sensitivity of LC-MS-MS to quantitate trace level components within complex matrices, in particular human plasma. One assay undergoes a validation procedure and demonstrates the utility of this new technology for drug quantitation within a regulated environment.

Bicyclic N-hydroxyurea inhibitors of 5-lipoxygenase: pharmacodynamic, pharmacokinetic, and in vitro metabolic studies characterizing N-hydroxy-N-(2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl)urea.

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.

Metabolism of the leukotriene receptor antagonist 5-(2-(8-phenyloctyl)phenyl)-4,6-dithianonanedioic acid (SK&F 102922) in the guinea pig. Rearrangement of the acyl glucuronide.

A primary route of inactivation of leukotrienes and their receptor antagonists (LTRA) is metabolism by omega oxidation. SK&F 102922 [5-(2-(8-phenyloctyl)phenyl)-4,6-dithianonanedioic acid] is a LTRA that was designed to be resistant to omega oxidation. Therefore, these experiments were designed to characterize the metabolic fate of [14C]SK&F 102922. Following iv administration of SK&F 102922 (5 mg/kg), 80% of injected radioactivity was excreted in bile in 1 hr. At least five metabolites and parent (18% of administered dose) were present in bile. One metabolite (M1), which accounted for less than 10% of the excreted radioactivity, was monohydroxylated. Three metabolites (M2, M3A, and M3B), which together accounted for greater than 50% of excreted radioactivity, had mass spectra consistent with acyl glucuronides. All three metabolites were alkali labile, whereas only one metabolite (M2) was susceptible to beta-glucuronidase hydrolysis. These data indicate that M3a and M3b are nonglycosidic isomers of M2 that were formed by a nonenzymic reaction involving migration of the aglycone (SK&F 102922) from C-1 to C-2, C-3, or C-4 of glucuronic acid. The 1-O-acyl-beta-glucuronide of SK&F 102922 (M2) exhibits pH dependent rearrangement, with half-lives ranging from 1 to greater than 1000 hr. Therefore, acyl glucuronidation can account for much of the metabolic fate of SK&F 102922 and, potentially, other structurally related LTRAs or endogenous leukotrienes themselves.

Comparative biochemical and morphometric changes associated with induction of the hepatic mixed function oxidase system in the rat.

This study characterized the induction of the rat hepatic cytochrome P-450-dependent mixed function oxidase system by SK&F 86002 [6-(4'-fluorophenyl)-5-(4'-pyridyl)-2,3-dihydroimidazo-(2,1-b)thia zole], an inhibitor of both the cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism. The induction characteristics of SK&F 86002 were compared to those of the classical inducer, phenobarbital, and morphological features of both SK&F 86002 and phenobarbital induced hepatocellular hypertrophy were quantitated. Rats were administered either SK&F 86002 (6, 18, or 60 mg/kg/day, po) or phenobarbital (8, 24, 80 mg/kg/day, ip) for 3 or 14 consecutive days. Liver to body weight ratio, total hepatic microsomal protein and cytochrome P-450 content, ethoxycoumarin-O-deethylase (ECOD) and leukotriene B4(LTB4) omega- and omega-1 hydroxylase were measured. Ultrastructural morphometry of the liver from control, and high dose SK&F 86002 (60 mg/kg/day) and phenobarbital (80 mg/kg/day) treated rats was completed. On day 3, phenobarbital increased liver to body weight ratio but only at the 80 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity increased in a dose-dependent fashion. LTB4 omega- and omega-1 hydroxylase activities were unaffected. Administration of SK&F 86002 for 3 days increased the liver to body weight ratio at both the 18 and 60 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity was significantly increased by the 60 mg/kg/day dosages of SK&F 86002. On day 14, phenobarbital increased the liver to body weight ratio and microsomal protein content but again only at the 80 mg/kg/day dosage. Cytochrome P-450 content was increased by all dosages.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of the novel antiinflammatory agent 6-(4'-fluorophenyl)-5-(4'-pyridyl)-2,3-dihydroimidazo-[2,1-b]-thiazole.

6-(4'-Fluorophenyl)-5-(4'-pyridyl)-2,3-dihydroimidazo-[2,1-b]-thia zole (SK&F 86002) is a dual inhibitor of arachidonic acid metabolism which has therapeutic potential for the treatment of inflammatory diseases. Previous studies in rats, in vivo, demonstrated that SK&F 86002 metabolism proceeds by sequential steps of sulfur and nitrogen oxidation. Therefore, these studies were designed to 1) identify the enzymes (flavin vs. cytochrome P-450-dependent monooxygenases) which were responsible for SK&F 86002 metabolism in vitro in hepatic microsomal suspensions from Sprague-Dawley rats, 2) characterize sex-dependent differences, and 3) quantitate the effect of pretreatment with SK&F 86002. All three steps in the sequential metabolism of SK&F 86002 to the N-oxide sulfone metabolite were quantitated individually. The three oxidation steps appeared to be catalyzed primarily by cytochrome P-450; heat inactivation (used to destroy flavin monooxygenase) had little effect on the metabolism of each compound. Further,N-octylamine failed to stimulate the metabolism of any compound and the cytochrome P-450 inhibitors (SK&F 525-A, metyrapone, and alpha-naphthoflavone) resulted in a marked inhibition of the metabolism of all three substrates. Maximal velocities for metabolism of all three substrates (SK&F 86002, sulfoxide, and sulfone) in microsomes isolated from male rats, were 3- to 5-fold greater than observed in female rats. Furthermore, pretreatment of male rats with SK&F 86002 (60 mg/kg/day for 3 days) resulted in a change in the in vitro metabolism of all three substrates generally characterized by an increase in Vmax and/or a fall in km.(ABSTRACT TRUNCATED AT 250 WORDS)

SK&F S-106203 inhibits leukotriene C4, leukotriene D4 and leukotriene E4 vasopressor responses in the conscious rat.

1. The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F S-106203, on leukotriene C4 (LTC4), LTD4 and LTE4 vasopressor responses in the conscious, normotensive rat. SK&F S-106203 was administered as a bolus followed by a continuous infusion in order to provide information on the relationship between antagonism of leukotriene responses and steady-state plasma concentrations. 2. Infusion of SK&F S-106203 at doses of 0.2 mgkg-1 + 1 mgkg-1 h-1, 1 mgkg-1 + 3 mgkg- h-1 or 2 mgkg-1 + 10 mgkg-1 h-1 produced dose-dependent steady-state plasma drug concentrations of 1.0, 3.2 and 23.8 micrograms ml-1, respectively. Plasma SK&F S-106203 concentrations appeared to increase in a linear fashion at doses of 1 and 3 mgkg-1 h-1; at the highest dose the increment in plasma drug concentrations (i.e., 7-8 fold) was greater than the increment in dose (i.e., 3 fold), suggesting saturation of the primary clearance mechanism(s) at this dose. 3. SK&F S-106203 (2 mgkg-1 + 10 mgkg-1 h-1) had no effect on noradrenaline-, vasopressin-, isoprenaline-, or U 46619-induced responses. 4. SK&F S-106203 produced dose-dependent rightward shifts in the LTC4 and LTE4 dose-response curves. Administration of SK&F S-106203 at doses of 0.2mg kg1 + 1 mg kg1 h-, mg kg' + 3mgkg-'h-1, or 2mgkg-' + lOmgkg-1h'- produced dose-ratios of 1.0, 3.1 and 19.9, respectively, against LTC4 responses, and dose-ratios of 1.6, 3.8 and 9.1, respectively, against LTE4 responses. 5. Against LTD4 responses, SK&F S-106203 at doses of 0.2mgkg- + mgkg-1 h-, mg kg' + 3 mg kg- 1h - ', or 2 mg kg- + 10 mg kg- h- produced dose-ratios of 2.5, 2.8, and 11.4, respectively. Administration of D-penicillamine, a non-competitive LTD4 dipeptidase inhibitor, had no effect on LTD4 responses. 6. The similarity in the LTD4 dose-ratios at the two lower infusion rates, despite increases in the plasma drug concentrations, suggests the existence of pharmacologically heterogeneous LTD4 receptors. These results indicate that SK&F S-106203 is a potent, selective and apparently competitive antagonist of LTC4, LTD4 and LTE4 vascular responses in the intact rat.

Antagonism of leukotriene C4, leukotriene D4 and leukotriene E4 vasoconstrictor responses in the conscious rat with the peptidoleukotriene receptor antagonist SK&F 104353: evidence for leukotriene D4 receptor heterogeneity.

The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F 104353, on leukotriene (LT)C4, LTD4 and LTE4 vasopressor responses in conscious, normotensive rats. Steady-state plasma concentrations of SK&F 104353 at infusion rates of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr were 0.5, 1.6 and 9.4 micrograms/ml, respectively, indicating that the plasma concentrations of SK&F 104353 were related directly to the infusion rate. LTC4, LTD4 and LTE4 (0.17-170 nmol/kg i.v.) produced dose-dependent increases in mean blood pressure. The ED20 dose (i.e., dose required to increase blood pressure 20 mm Hg) of LTC4, LTD4 or LTE4 was 2.7 +/- 0.4, 2.2 +/- 0.3 and 109 +/- 17 nmol/kg, respectively. SK&F 104353 produced dose-dependent, parallel shifts to the right in the LTC4 dose-response curve. Administration of SK&F 104353 at doses of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr produced dose ratios (i.e., ratio of ED20 in presence of SK&F 104353 to that of the vehicle group) of 6, 12 and 26, respectively. Against LTD4 responses, SK&F 104353 at doses of 0.1 mg/kg + 0.3 mg/kg/hr or 0.2 mg/kg + 1 mg/kg/hr produced dose ratios of 3 and 9, respectively. At a dose of 1 mg/kg + 3 mg/kg/hr, there was no further increase in the dose ratio, whereas a dose of 2 mg/kg + 10 mg/kg/hr resulted in a dose ratio of greater than 100.(ABSTRACT TRUNCATED AT 250 WORDS)

The bronchopulmonary pharmacology of SK&F 104353 in anesthetized guinea pigs: demonstration of potent and selective antagonism of responses to peptidoleukotrienes.

The bronchopulmonary pharmacology of SK&F 104353 [2(S)-hydroxy-3(R)-[2(2-carboxyethyl)thio]-3[2-(8- phenyloctyl)phenyl]-propanoic acid], a potent and selective leukotriene (LT) receptor antagonist in vitro, was assessed in anesthetized, spontaneously breathing guinea pigs. Aerosol administration of SK&F 104353 (5-2000 micrograms/ml x 100 breaths) reduced in a concentration-dependent manner the response to a standard LTD4 challenge (4.33 micrograms/ml x 5 breaths) given 30 min later. Inhalation of a 2000 micrograms/ml solution abolished LTD4-induced bronchoconstriction for at least 2 hr. The i.v. administration of SK&F 104353 10 min before challenge antagonized LTD4-induced bronchoconstriction with an ID50 of 0.55 mumol/kg (0.25 mg/kg). Substantial antagonism of LTD4-induced bronchospasm was observed for at least 60 min after i.v. administration of 5 mumol/kg of SK&F 104353. Infusion of SK&F 104353 at various rates revealed that a steady-state plasma concentration of 0.125 microM (0.06 micrograms/ml) reduced LTD4-induced bronchoconstriction by 60%. In addition to preventing the response to LTD4, i.v. administered SK&F 104353 (10 mumol/kg) rapidly and completely reversed ongoing LTD4-induced bronchoconstriction. SK&F 104353 also was effective when given intraduodenally 1 hr before LTD4 challenge, although the ID50 (32 mumol/kg) was 60-fold greater than the i.v. ID50. Given intragastrically, 100 mumol/kg of SK&F 104353 abolished the response to LTD4 for 1 hr, and reduced the response for 6 hr. SK&F 104353 (20 mumol/kg i.v.) had no effect on the bronchoconstriction induced by aerosolized acetylcholine, histamine or U-44069, but did antagonize the response to LTC4. SK&F 104353 alone did not produce bronchoconstriction when administered by any route or dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective antagonism of peptidoleukotriene responses does not reduce myocardial damage or neutrophil accumulation following coronary artery occlusion with reperfusion.

This study was designed to assess the effect of a peptidoleukotriene receptor antagonist, SK&F 104353, for limiting myocardial damage and neutrophil accumulation in rats subjected to myocardial reperfusion injury (MI/R). In conscious rats, SK&F 10,4353 (25 mg/kg, i.v.) antagonized LTD4-induced vasopressor responses by 90% and 60% at 1 and 4 hr, respectively, indicating effective blockade of peptido-leukotriene responses. In another group of animals subjected to 30 min of coronary artery occlusion with reperfusion for 24 hr, myocardial injury and neutrophil infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the left ventricular free wall (LVFW). Myocardial CPK levels were 8.1 +/- 0.2 U/mg protein in Sham-MI/R vehicle-treated animals, and were significantly decreased to 6.4 +/- 0.6 U/mg protein in MI/R-vehicle animals. Myocardial MPO values were 1.5 +/- 0.5 U/g LVFW in Sham-MI/R vehicle-treated animals, and significantly increased to 4.3 +/- 0.6 U/g LVFW in MI/R-vehicle animals. Administration of SK&F 10,4353 (25 mg/kg, i.v.) 1 min prior to coronary occlusion and 3.5 hr post reperfusion had no effect on the loss of myocardial CPK specific activity or the increase in MPO levels (p greater than 0.05, compared to the MI/R-vehicle group). Thus, at a dose that antagonized LTD4-induced vasopressor responses, SK&F 104353 did not attenuate either the extent of myocardial injury or inflammatory cell accumulation associated with myocardial ischemia/reperfusion. These results suggest that peptidoleukotrienes do not contribute to the progression of myocardial ischemic/reperfusion injury.

Pharmacokinetics and metabolism of SK&F 86002 in male and female Sprague-Dawley rats.

Several pharmacokinetic parameters for SK&F 86002 [6-(4'-fluorophenyl)-5-(4'-pyridyl)-2,3-dihydroimidazo(2,1-b)-thia zole] and its metabolites (sulfoxide, sulfone) were measured in male and female Sprague-Dawley rats after iv (5 mg/kg) and a wide range (10-80 mg/kg) of oral doses of SK&F 86002. In both sexes. SK&F 86002 is metabolized to an active metabolite, sulfone, which has an extended half-life (approximately 13 hr) and, therefore, has the potential to accumulate upon repeated dosing. In addition, striking differences between sexes were noted in several of the pharmacokinetic parameters measured. The AUCs areas under the plasma concentration time curves, for SK&F 86002 in female rats obtained at the higher doses of SK&F 86002 were substantially greater than expected, when compared with lower doses in female rats or with equivalent doses in male rats. Furthermore, at all doses of SK&F 86002, AUCs for sulfoxide were substantially larger in female than in male rats. Consequently, the systemic exposure to SK&F 86002 and metabolites is substantially greater in female rats than in male rats. Therefore, extrapolation of the pharmacologic and toxicologic sequelae of SK&F 86002, observed at larger doses in female rats, to lower doses should be approached cautiously. Furthermore, the extended half-life of the pharmacologically active sulfone metabolite of SK&F 86002 suggests that this metabolite could accumulate on repeated daily dosing of SK&F 86002 and could, therefore, account for much of the chronic pharmacologic and toxicologic activity of SK&F 86002.

Concentration-dependent, stereoselective inhibition of the endotoxin-induced hemoconcentration in conscious rats with the peptidoleukotriene receptor antagonist SK & F 104353.

Endotoxemia is associated with increases in a number of humoral mediators including vasopressin, thromboxane and leukotrienes (LT), all of which may participate in the pathophysiologic responses to endotoxemia. Previous studies from our laboratory demonstrated that endotoxin-induced hemoconcentration was attenuated with a peptidoleukotriene receptor antagonist, SK & F 104353. The purpose of this study was to investigate further the mechanism of endotoxin-induced hemoconcentration. Injection of LTD4 (51 nmol/kg, i.v.) produced an increase in the hematocrit of conscious male Sprague-Dawley rats: administration of SK & F 104353 (2 mg/kg, i.v. + 10 mg/kg/h, i.v. infusion) blocked completely this response to exogenous LTD4. Injection of Salmonella enteritidis endotoxin (30 mg/kg, i.v.) increased the hematocrit from 41 +/- 1 vol% to 55 +/- 1 vol%. Following pretreatment with SK & F 104353 (2 mg/kg, i.v. + 10 mg/kg/h, i.v.), the hemoconcentration was attenuated to 46 +/- 1 vol% (p less than 0.01). Simultaneous determination of plasma drug concentrations over a range of doses indicated that inhibition of the hemoconcentration produced by SK & F 104353 was concentration-dependent (IC30 = 0.5 microgram/ml). The IC30 for the stereoisomer, SK & F 104373, was 50 micrograms/ml. The 5-lipoxygenase/cyclooxygenase inhibitors, SK & F 86002 and BW 775C, also attenuated the endotoxin-induced increase in hematocrit, whereas indomethacin, heparin, daltroban, or the selective V1 vasopressin receptor antagonist [d(CH2)5Tyr(Me)]AVP did not significantly affect the endotoxin-induced hemoconcentration. The endotoxin-induced hemoconcentration was inhibited in a concentration-dependent, stereoselective manner with a peptidoleukotriene receptor antagonist, and by 5-lipoxygenase inhibitors, indicating that this response is mediated by peptidoleukotrienes.

Inhibition by ranitidine of acetaminophen conjugation and its possible role in ranitidine potentiation of acetaminophen-induced hepatotoxicity.

Pretreatment with ranitidine (RA) potentiates the hepatotoxicity of acetaminophen (APAP) in male Fischer 344 rats. The present studies were undertaken to investigate the role of APAP metabolism in this potentiation. Administration of RA (50 mg/kg p.o.) to male Fischer 344 rats 30 min before [3H]APAP (750 mg/kg p.o.) increased the plasma concentrations of acetaminophen at 2 hr (193%) and 4 hr (277%) after APAP. Covalent binding of [3H]APAP-related material to hepatic macromolecules in RA-pretreated animals was similar to APAP alone values up to 12 hr after treatment; however, 24 hr after APAP, binding in the RA-pretreated animals was twice that observed in animals given [3H]APAP alone. Urinary excretion (0-24 hr) of APAP and APAP glucuronide were reduced in ranitidine-pretreated animals to 64 and 66% of control, respectively, indicating that in vivo RA altered APAP conjugation with glucuronic acid. APAP uridine diphosphoglucuronyltransferase activity in rat hepatic microsomes was competitively inhibited by RA (0.1-2 mM). The Ki apparent for RA inhibition of APAP uridine diphosphoglucuronyltransferase was 0.04 mM. In contrast, neither APAP nor 4-nitrophenol sulfotransferase activity in rat hepatic cytosol was inhibited by RA at concentrations up to 5 mM. Together, these results support the suggestion that RA-mediated alterations of APAP conjugation may explain the potentiation of APAP-induced hepatotoxicity by RA in rats.

Ovarian effects of SK&F 86002-A2 in the rat: site of action.

In a preliminary 30-day study, oral administration of SK&F 86002-A2, an inhibitor of prostaglandin and leukotriene synthesis, blocked ovulation and altered ovarian structure and hormone production in rats. The purpose of the present study was to determine if the locus of action of SK&F 86002-A2 for these effects was the ovary or some other site in the female reproductive system, using a number of experimental approaches. A single sc or intraovarian injection of SK&F 86002-A2 did not block spontaneous or gonadotropin-induced ovulation in proestrous rats, whereas indomethacin, a positive control, acutely disrupted the ovulatory process. Since neither route of administration blocked ovulation, integrated pituitary and ovarian events were not negatively affected by a single injection of SK&F 86002-A2 at doses which caused ovarian dysfunction when administered repeatedly for 30 days. In contrast to a single dose, oral administration of SK&F 86002-A2 to hypophysectomized rats for 2 weeks suppressed follicular growth and estradiol production in response to sc administration of pregnant mare serum gonadotropin. Although ovarian function was suppressed in hypophysectomized rats, LH surges induced by estradiol in ovariectomized rats were not affected by administration of SK&F 86002-A2 for 2 weeks. Thus, hypothalamic/pituitary dysfunction did not contribute to the ovarian effects of SK&F 86002 that occurred after repeated dosing. In conclusion, these results indicate that disruption of ovarian cycles by SK&F 86002-A2 is related to a direct effect on the ovary, and not to altered hypothalamic/pituitary function and LH release. Specifically, SK&F 86002-A2 may suppress the ovarian response to gonadotrophin, retarding follicular growth and estrogen production. The ovarian effects are consistent with a pharmacological expression of the inhibitory action of SK&F 86002-A2 on prostaglandin and leukotriene synthesis.

Beneficial effects of the peptidoleukotriene receptor antagonist, SK&F 104353, on the responses to experimental endotoxemia in the conscious rat.

The purpose of this study was to examine the effects of the peptidoleukotriene receptor antagonist, SK&F 104353, on the responses to endotoxin in conscious male Sprague-Dawley rats. Administration of Salmonella enteritidis endotoxin (30 mg/kg i.v.; LD90) resulted in a decrease in the number of circulating platelets, leukopenia, an increase in hematocrit, and 0% survival at 24 hr. Pretreatment with SK&F 104353 (1 mg/kg, i.v. bolus followed by 3 mg/kg/hr, i.v. infusion for 6 hr) 5 min before injection of endotoxin produced steady state plasma drug levels of 1.6 micrograms/ml in naive animals and levels of approximately 3.4 micrograms/ml in endotoxemic animals (P less than 0.05). SK&F 104353 significantly attenuated the endotoxin-induced thrombocytopenia (P less than 0.05) but had no effect on either the endotoxin-induced early leukopenia or late leukocytosis. Additionally, SK&F 104353 significantly reduced the endotoxin-induced hemoconcentration (P less than 0.05) and improved survival to 30% at 48 hr (P less than 0.05). A higher dose of SK&F 104353 (2 mg/kg, i.v. bolus followed by 10 mg/kg/hr, i.v. infusion for 6 hr) did not produce any further benefit. These data indicate clearly the pathophysiologic role of peptidoleukotrienes in endotoxemia and suggest SK&F 104353 could be useful for ameliorating some of the deleterious sequelae associated with this condition.

Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation.

An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, alpha-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation [omega-, (omega-1)-, (omega-2)-] of LA may be catalyzed by different isozymes of cytochrome P-450.

In vitro microsomal metabolism of the leukotriene receptor antagonist, 5-(2-dodecylphenyl)-4,6-dithianonanedioic acid (SK&F 102,081).

In vivo experiments indicate that the major route of metabolism of SK&F 102,081 [5-(2-dodecylphenyl)-4,6-dithianonanedioic acid] is via omega-oxidation and subsequent beta-oxidation. Therefore, in vitro experiments were designed to characterize the initial reaction of this pathway, omega-hydroxylation. SK&F 102,081 was metabolized by rat hepatic microsomes to two products; mass spectral analysis indicated that these products were the omega (omega) and omega minus one (omega-1) hydroxylated metabolites, 5-[2-(12-hydroxy)dodecylphenyl]-4,6-dithianonanedioic acid and 5-[2-(11-hydroxy)dodecylphenyl]-4,6-dithianonanedioic acid, respectively. NADPH and oxygen were required for the formation of these metabolites. Kinetic analysis of omega- and (omega-1)-hydroxylations of SK&F 102,081 indicated that the apparent Km for (omega-1)-hydroxylation (52.6 microM) was approximately 2.5-fold higher than the apparent Km for omega-hydroxylation (22.0 microM). Furthermore, the cytochrome P-450 inhibitor, metyrapone, produced differential inhibition of omega- and (omega-1)-hydroxylation. In addition, the terminal acetylenic analogue of SK&F 102,081, SK&F 103,600 (5-[2-(11-dodecynyl)phenyl]-4,6-dithianonanedioic acid), produced differential suicide inactivation of SK&F 102,081 omega- and (omega-1)-hydroxylations. These studies indicate that the omega- and (omega-1)-hydroxylations of SK&F 102,081 are probably carried out by different isozymes of hepatic cytochrome P-450 in the rat. Furthermore, the isozymes that hydroxylate SK&F 102,081 at the omega- and (omega-1)-positions may be similar to those which mediate similar reactions on endogenous compounds such as prostaglandins and leukotrienes.

In vivo metabolism of the leukotriene receptor antagonist, 5-(2-dodecylphenyl)-4,6-dithianonanedioic acid (SK&F 102,081) in the guinea pig.

Both leukotrienes and their receptor antagonists possess substantial pharmacologic activity in in vitro systems, but their duration of action in vivo is extremely short. The exact mechanism of rapid inactivation of these lipids is unknown, but is likely due to metabolism. Therefore, the metabolic fate of a model antagonist 5-(2-dodecylphenyl)-4,6-dithianonanedioic acid (SK&F 102,081) was elucidated in anesthetized guinea pigs. Following iv administration of [14C]SK&F 102,081 (5 mg/kg), 85% of injected radioactivity was excreted in bile in 1 hr. Approximately 6% of the radioactivity in bile was associated with parent. At least 14 metabolites were present in bile, 2 of which accounted for almost 60% of the excreted radioactivity. Identification of biliary metabolites revealed that metabolism occurred by two major routes, omega-oxidation with subsequent beta-oxidation and acyl glucuronidation at approximately a 4:1 ratio. Since current structure-activity relationships suggest that omega-oxidation results in the loss of pharmacologic activity of SK&F 102,081, the rapid loss in pharmacologic activity observed in vivo may be due to rapid metabolism.

Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases.

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.